l-2-Hydroxyglutarate contributes to tumor radioresistance through regulating the hypoxia-inducible factor-1α signaling pathway.

l-2-Hydroxyglutarate (l-2-HG) has been regarded as a tumor metabolite, and it plays a crucial role in adaptation of tumor cells to hypoxic conditions. However, the role of l-2-HG in tumor radioresistance and the underlying mechanism have not yet been revealed. Here, we found that l-2-HG exhibited to have radioresistance effect on U87 human glioblastoma cells, which could reduce DNA damage and apoptosis caused by irradiation, promote cell proliferation and migration, and impair G2/M phase arrest. Mechanistically, l-2-HG upregulated the protein level of hypoxia-inducible factor-1α (HIF-1α) and the expression levels of HIF-1α downstream target genes. The knockdown of l-2-hydroxyglutarate dehydrogenase (L2HGDH) gene promoted the tumor growth and proliferation of U87 cells in nude mice by increasing HIF-1α expression level in vivo. In addition, the low expression level of L2HGDH gene was correlated with the short survival of patients with glioma or kidney cancer. In conclusion, our study revealed the role and mechanism of l-2-HG in tumor radioresistance and may provide a new perspective for overcoming tumor radioresistance and broaden our comprehension of the role of metabolites in tumor microenvironment.

Inhibition of ERN1 affects the expression of TGIF1 and other homeobox gene expressions in U87MG glioblastoma cells.

BACKGROUND: The ERN1 (endoplasmic reticulum to nucleus signaling 1) pathway plays an important role in the regulation of gene expression in glioblastoma, but molecular mechanism has not yet been fully elucidated. The aim of this study was to evaluate the relative relevance of ERN1 activity as a kinase in comparison to its endoribonuclease activity in the regulation of homeobox gene expression. METHODS: Two sublines of U87MG glioblastoma cells with different ways of ERN1 inhibition were used: dnERN1 (overexpressed transgene without protein kinase and endoribonuclease) and dnrERN1 (overexpressed transgene with mutation in endoribonuclease). ERN1 suppression was also done using siRNA for ERN1. Silencing of XBP1 mRNA by specific siRNA was used for suppression of ERN1 endoribonuclease function mediated by XBP1s. The expression levels of homeobox genes and microRNAs were evaluated by qPCR. RESULTS: The expression of TGIF1 and ZEB2 genes was downregulated in both types of glioblastoma cells with inhibition of ERN1 showing the ERN1 endoribonuclease-dependent mechanism of their regulation. However, the expression of PBX3 and PRPRX1 genes did not change significantly in dnrERN1 glioblastoma cells but was upregulated in dnERN1 cells indicating the dependence of these gene expressions on the ERN1 protein kinase. At the same time, the changes in PAX6 and PBXIP1 gene expressions introduced in glioblastoma cells by dnrERN1 and dnERN1 were different in direction and magnitude indicating the interaction of ERN1 protein kinase and endoribonuclease activities in regulation of these gene expressions. The impact of ERN1 and XBP1 silencing on the expression of studied homeobox genes is similar to that observed in dnERN1 and dnrERN1 glioblastoma cells, correspondingly. CONCLUSION: The expression of TGIF1 and other homeobox genes is dependent on the ern1 signaling pathways by diverse mechanisms because inhibition of ERN1 endoribonuclease and both ERN1 enzymatic activities had dissimilar impacts on the expression of most studied genes showing that ERN1 protein kinase plays an important role in controlling homeobox gene expression associated with glioblastoma cell invasion.

Telmisartan loaded lipid nanocarrier as a potential repurposing approach to treat glioma: characterization, apoptosis evaluation in U87MG cells, pharmacokinetic and molecular simulation study.

The study explores anticancer potential of telmisartan (TS) loaded lipid nanocarriers (TLNs) in glioma cells as a potential repurposing nanomodality along with estimation of drug availability at rat brain. Experimental TLNs were produced by previously reported method and characterized.In vitroanticancer efficacy of experimental TLNs was estimated by MTT, confocal microscopy, and FACs analysis in glioma cells. Plasma and brain pharmacokinetic (PK) parameters were also analysed by LCMS/MS. Spherical, nanosized, homogenous, unilamellar, TLNs were reported having desirable drug loading (9.5% ± 0.6%), negative zeta potential and sustained TS release tendency. FITC-TLNs were sufficiently internalized into U87MG cells line within 0.5 h incubation period. IC50for TLNs was considerably higher than free TS in the tested glioma cell lines. Further, TLNs induced superior apoptotic effect in U87MG cells than TS. PK (plasma/brain) data depicted higher AUC,Vss, MRT with lower Cltfor TLNs suggesting improved bioavailability,in vivoresidence and sustained drug availability than free TS administration. Docking studies rationalizedin vitro/in vivoresults as preferably higher binding affinity (docking score:12.4) was detected for TS with glioma proteins. Further,in vivostudies in glioma bearing xenograft model is underway for futuristic clinical validation of TLNs.

Endoplasmic reticulum stress-dependent regulation of the expression of serine hydroxymethyltransferase 2 in glioblastoma cells.

Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.

ERN1 knockdown modifies the hypoxic regulation of homeobox gene expression in U87MG glioblastoma cells.

OBJECTIVE.: Homeobox genes play an important role in health and disease including oncogenesis. The present investigation aimed to study ERN1-dependent hypoxic regulation of the expression of genes encoding homeobox proteins MEIS (zinc finger E-box binding homeobox 2) and LIM homeobox 1 family, SPAG4 (sperm associated antigen 4) and NKX3-1 (NK3 homeobox 1) in U87MG glioblastoma cells in response to inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioblastoma growth. METHODS.: The expression level of homeobox genes was studied in control (transfected by vector) and ERN1 knockdown U87MG glioblastoma cells under hypoxia induced by dimethyloxalylglycine (0.5 mM for 4 h) by quantitative polymerase chain reaction and normalized to ACTB. RESULTS.: It was found that hypoxia down-regulated the expression level of LHX2, LHX6, MEIS2, and NKX3-1 genes but up-regulated the expression level of MEIS1, LHX1, MEIS3, and SPAG4 genes in control glioblastoma cells. At the same time, ERN1 knockdown of glioblastoma cells significantly modified the sensitivity of all studied genes to a hypoxic condition. Thus, ERN1 knockdown of glioblastoma cells removed the effect of hypoxia on the expression of MEIS1 and LHX1 genes, but increased the sensitivity of MEIS2, LHX2, and LHX6 genes to hypoxia. However, the expression of MEIS3, NKX3-1, and SPAG4 genes had decreased sensitivity to hypoxia in ERN1 knockdown glioblastoma cells. Moreover, more pronounced changes under the conditions of ERN1 inhibition were detected for the pro-oncogenic gene SPAG4. CONCLUSION.: The results of the present study demonstrate that hypoxia affected the expression of homeobox genes MEIS1, MEIS2, MEIS3, LHX1, LHX2, LHX6, SPAG4, and NKX3-1 in U87MG glioblastoma cells in gene-specific manner and that the sensitivity of all studied genes to hypoxia condition is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling, and possibly contributed to the control of glioblastoma growth. A fundamentally new results of this work is the establishment of the fact regarding the dependence of hypoxic regulation of SPAG4 gene expression on ER stress, in particular ERN1, which is associated with suppression of cell proliferation and tumor growth.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

Exploring the Neuroprotective Potential of N-Methylpyridinium against LPS-Induced Neuroinflammation: Insights from Molecular Mechanisms.

N-methylpyridinium (NMP) is produced through the pyrolysis of trigonelline during the coffee bean roasting process. Preliminary studies suggest that NMP may have health benefits, thanks to its antioxidant properties. Based on this background, the aim of this study was to evaluate whether NMP could have a protective effect against LPS-induced neuroinflammation in human glioblastoma cells (U87MG). With this aim, U87MG cells were pre-treated with NMP (0.5 µM) for 1 h and then exposed to LPS (1 µg/mL) for 24 h. Our findings show that NMP attenuates LPS-induced neuroinflammation by reducing the expression of pro-inflammatory cytokines, such as IL-1ß, TNF-α and IL-6, through the inhibition of the NF-κB signaling pathway, which is critical in regulating inflammatory responses. NMP is able to suppress the activation of the NF-κB signaling pathway, suggesting its potential in preventing neuroinflammatory conditions. These outcomes support the notion that regular consumption of NMP, possibly through coffee consumption, may offer protection against neuroinflammatory states implicated in neurological disorders.

Transmembrane Protein TMEM230, Regulator of Glial Cell Vascular Mimicry and Endothelial Cell Angiogenesis in High-Grade Heterogeneous Infiltrating Gliomas and Glioblastoma.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

In vivo antiangiogenic effect of nimbolide, trans-chalcone and piperine for use against glioblastoma.

BACKGROUND: Angiogenesis is an important hallmark of Glioblastoma (GBM) marked by elevated vascular endothelial growth factor-A (VEGF-A) and its receptor 2 (VEGFR-2). As previously reported nimbolide (NBL), trans-chalcone (TC) and piperine (PPR) possess promising antiangiogenic activity in several cancers however, their comparative efficacy and mechanism of antiangiogenic activity in GBM against VEGFR-2 has not been elucidated. METHODS: 2D and 3D spheroids cultures of U87 (Uppsala 87 Malignant Glioma) were used for evaluation of non-cytotxoic dose for anti-angiogenic activity. The antiangiogenic effect was investigated by the GBM U87 cell line bearing chick CAM model. Excised U87 xenografts were histologically examined for blood vascular density by histochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of avian and human VEGF-A and VEGFR-2 mRNA transcripts. RESULTS: Using 2D and 3D spheroid models, the non-cytotoxic dose of NBL, TC and PPR was ≤ 11 µM. We found NBL, TC and PPR inhibit U87-induced neoangiogenesis in a dose-dependent manner in the CAM stand-alone model as well as in CAM U87 xenograft model. The results also indicate that these natural compounds inhibit the expression of notable angiogenic factors, VEGF-A and VEGFR-2. A positive correlation was found between blood vascular density and VEGF-A as well as VEGFR-2 transcripts. CONCLUSION: Taken together, NBL, TC and PPR can suppress U87-induced neoangiogenesis via a reduction in VEGF-A and its receptor VEGFR-2 transcript expression at noncytotoxic concentrations. These phytochemicals showed their utility as adjuvants to GBM therapy, with Piperine demonstrating superior effectiveness among them all.

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG.

BACKGROUND: HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model. METHODS: Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry. RESULTS: The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG. CONCLUSIONS: We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.

Synthesis, biological evaluation and molecular docking studies of novel 1,3,4-thiadiazoles as potential anticancer agents and human carbonic anhydrase inhibitors.

Thiosemicarbazide and also 1,3,4-thiadiazole derivatives have been garnering substantial attention from researchers worldwide due to their expansive range of biological activities, encompassing antimicrobial, anti-inflammatory, and anticancer properties. Herein, we embarked on a comprehensive investigation in this study, introducing a novel series of thiosemicarbazides (3a-3i) and their corresponding 1,3,4-thiadiazole (4a-4i) derivatives. The compounds were meticulously designed, synthesized, and subjected to meticulous characterization using various spectroscopic methods such as FT-IR, 1H-NMR, 13C-NMR, and elemental analysis. Afterward, their potential anti-proliferative effectiveness was assessed using MTT assay against two cancer cell lines (U87 and HeLa) and normal fibroblast cells (L929). Among the compounds, 4d showed the highest cytotoxic activity against U87 and 4i against HeLa. Compound 3b exhibited selective cytotoxic activity against both cancer cells. Among the molecules with selective activity against the U87 cell line; 3a, 3b, 4d and 4e were further evaluated by caspase-3 activity levels, Bax and Bcl-2 protein expression, and total oxidant status assay. Besides, carbonic anhydrase IX activity studies were also performed in order to understand the underlying mechanism of action. The results indicated that compound 4e showed higher efficacy than standard acetazolamide (IC50 = 0.58 ± 0.02 µM) with an IC50 value of 0.03 ± 0.01 µM. Furthermore, molecular docking studies were carried out using carbonic anhydrase IX crystals to determine the compound's interactions with the enzyme's active sites. This comprehensive investigation sheds light on the intricate interplay between molecular structure and biological activity, providing valuable insights into the therapeutic potential of these compounds.

ERN1 dependent impact of glucose and glutamine deprivations on PBX3, PBXIP1, PAX6, MEIS1, and MEIS2 genes expression in U87 glioma cells.

Objective. Homeobox genes play a fundamental role in the embryogenesis, but some of them have been linked to oncogenesis. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of homeobox genes such as PAX6 (paired box 6), PBX3 (PBX homeobox 3), PBXIP1 (PBX homeobox interacting protein 1), MEIS1 (MEIS homeobox 1), and MEIS2 in ERN1 knockdown U87 glioma cells with the intent to reveal the role of ERN1 (endoplasmic reticulum to nucleus signaling 1) signaling pathway on the endoplasmic reticulum stress dependent regulation of homeobox genes. Methods. The control (transfected by empty vector) and ERN1 knockdown (transfected by dominant-negative ERN1) U87 glioma cells were exposed to glucose and glutamine deprivations for 24 h. The cells RNA was extracted and reverse transcribed. The expression level of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes was evaluated by a real-time quantitative polymerase chain reaction analysis and normalized to ACTB. Results. It was found that glucose deprivation down-regulated the expression level of PAX6, MEIS1, and MEIS2 genes in control glioma cells, but did not significantly alter PBX3 and PBXIP1 genes expression. At the same time, ERN1 knockdown significantly modified the sensitivity of all studied genes to glucose deprivation. Other changes in gene expression were detected in control glioma cells under the glutamine deprivation. The expression of PBX3 and MEIS2 genes was down- while PAX6 and PBXIP1 genes up-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on the majority of studied genes expression in U87 glioma cells. Conclusion. The results of the present study demonstrate that the exposure of U87 glioma cells under glucose and glutamine deprivations affected the expression of the majority of the studied homeobox genes and that the sensitivity of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes expression under these experimental conditions is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling.

Expression of DNAJB9 and some other genes is more sensitive to SWCNTs in normal human astrocytes than glioblastoma cells.

Objective. Single-walled carbon nanotubes (SWCNTs) are considered to be one of the nanomaterials attractive for biomedical applications, particularly in the health sciences as imaging probes and drug carriers, especially in the field of cancer therapy. The increasing exploitation of nanotubes necessitates a comprehensive evaluation of the potential impact of these nanomaterials, which purposefully accumulate in the cell nucleus, on the human health and the function of the genome in the normal and tumor tissues. The aim of this study was to investigate the sensitivity of the expression of DNAJB9 and some other genes associated with the endoplasmic reticulum (ER) stress and cell proliferation to low doses of SWCNTs in normal human astrocytes (NHA/TS) and glioblastoma cells (U87MG) with and without an inhibition of ERN1 signaling pathway of the ER stress. Methods. Normal human astrocytes, line NHA/TS and U87 glioblastoma cells stable transfected by empty vector or dnERN1 (dominant-negative construct of ERN1) were exposed to low doses of SWCNTs (2 and 8 ng/ml) for 24 h. RNA was extracted from the cells and used for cDNA synthesis. The expression levels of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 mRNAs were measured by a quantitative polymerase chain reaction and normalized to ACTB mRNA. Results. It was found that the low doses of SWCNTs up-regulated the expression of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, CLU, and P4HA2 genes in normal human astrocytes in dose-dependent (2 and 8 ng/ml) and gene-specific manner. These nanotubes also increased the expression of most studied genes in control (transfected by empty vector) U87 glioblastoma cells, but with much lesser extent than in NHA/TS. However, the expression of CLU gene in control U87 glioblastoma cells treated with SWCNTs was down-regulated in a dose-dependent manner. Furthermore, the expression of TOB1 and P4HA2 genes did not significantly change in these glioblastoma cells treated by lower dose of SWCNTs only. At the same time, inhibition of ERN1 signaling pathway of ER stress in U87 glioblastoma cells led mainly to a stronger resistance of DNAJB9, TOB1, BRCA1, DDX58, TFPI2, and P4HA2 gene expression to both doses of SWCNTs. Conclusion. The data obtained demonstrate that the low doses of SWCNTs disturbed the genome functions by changing the levels of key regulatory gene expressions in gene-specific and dose-dependent manner, but their impact was much stronger in the normal human astrocytes in comparison with the tumor cells. It is possible that ER stress, which is constantly present in tumor cells and responsible for multiple resistances, also created a partial resistance to the SWCNTs action. Low doses of SWCNTs induced more pronounced changes in the expression of diverse genes in the normal human astrocytes compared to glioblastoma cells indicating for a possible both genotoxic and neurotoxic effects with a greater extent in the normal cells.

Hypoxia controls the expression of genes responsible for serine synthesis in U87MG cells on ERN1-dependent manner.

Objective. Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling) significantly suppressed the glioblastoma cell proliferation and modified the hypoxia regulation. The present study is aimed to investigate the impact of hypoxia on the expression of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine aminotransferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) in U87MG glioblastoma cells in relation to knockdown of ERN1 with the intent to reveal the role of ERN1 signaling pathway on the endoplasmic reticulum stress-dependent regulation of expression of these genes. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed to hypoxia introduced by dimethyloxalylglycine for 4 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH, PSAT1, PDPH, SHMT1, and ATF4 genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that hypoxia up-regulated the expression level of PHGDH, PSAT1, and ATF4 genes in control U87MG cells, but PSPH and SHMT1 genes expression was down-regulated. The expression of PHGDH, PSAT1, and ATF4 genes in glioblastoma cells with knockdown of ERN1 signaling protein was more sensitive to hypoxia, especially PSAT1 gene. At the same time, the expression of PSPH gene in ERN1 knockdown cells was resistant to hypoxia. The expression of SHMT1 gene, encoding the enzyme responsible for conversion of serine to glycine, showed similar negative sensitivity to hypoxia in both control and ERN1 knockdown glioblastoma cells. Conclusion. The results of the present study demonstrate that the expression of genes responsible for serine synthesis is sensitive to hypoxia in gene-specific manner and that ERN1 knockdown significantly modifies the impact of hypoxia on the expression of PHGDH, PSAT1, PSPH, and ATF4 genes in glioblastoma cells and reflects the ERN1-mediated reprograming of hypoxic regulation at gene expression level.

Capsaicin induces redox imbalance and ferroptosis through ACSL4/GPx4 signaling pathways in U87-MG and U251 glioblastoma cells.

Glioblastoma is one of the deadliest malignant gliomas. Capsaicin is a homovanillic acid derivative that can show anti-cancer effects by regulating various signaling pathways. The aim of this study is to investigate the effects of capsaicin on cell proliferation via ferroptosis in human U87-MG and U251 glioblastoma cells. Firstly, effects of capsaicin treatment on cell viability were determined by MTT analysis. Next, cellular-proliferation and cytotoxicity assays were determined by analyzing bromodeoxyuridine (BrdU) and lactate dehydrogenase (LDH) activity, respectively. Following, acyl-CoA synthetase long chain family member 4 (ACSL4), glutathione peroxidase 4 (GPx4), 5-hydroxyeicosatetraenoic acid (5-HETE), total oxidant status (TOS), malondialdehyde (MDA), total antioxidant status (TAS) and reduced glutathione (GSH) levels were determined by ELISA. Additionally, ACSL4 and GPx4 mRNA and protein levels were analyzed. Capsaicin showed a concentration-dependent anti-proliferative effects in U87-MG and U251 cells. Cell viability was decreased in the both cell lines treated with capsaicin concentrations above 50 µM, while LDH activity increased. Treatment of 121.6, 188.5, and 237.2 µM capsaicin concentrations for 24 h indicated an increase in ACSL4, 5-HETE, TOS and MDA levels in U87-MG and U251 cells (p < 0.05). On the other hand, we found that capsaicin administration caused a decrease in BrdU, GPx4, TAS and GSH levels in U87-MG and U251 cells (p < 0.05). Besides, ACSL4 mRNA and protein levels were increased in the glioblastoma cells treated with capsaicin, whereas GPx4 mRNA and protein levels were decreased. Finally, capsaicin might be used as a potential anticancer agent with ferroptosis-induced anti-proliferative effects in the treatment of human glioblastoma.

Novel Chalcone-Phenazine Hybrids Induced Ferroptosis in U87-MG Cells through Activating Ferritinophagy.

Thirty-seven novel chalcone-phenazine hybrid molecules (C1∼C13 and F1∼F24) with 1,2,3-triazole or ethyl group as linkers were designed and synthesized in this study. Some compounds exhibited selective cytotoxicity against U87-MG cancer cell lines in vitro, in which compound C4 were found to have the best antiproliferative activity. SAR study indicated 1,2,3-triazole group may be crucial for enhancing compounds' cytotoxicity. C4 was verified to induce ferroptosis in U87-MG cells by transcription, lipid peroxidation, lipid ROS assays. Furthermore, C4 was up-regulated LC3-II, degradated FTH1, and then increasing iron resulted in the down-regulation of NCOA4. Together, all above evidences highlighted the potential of compound C4 that triggered ferroptosis by activating ferritinophagy against U87-MG cells.

Anti-Proliferative Potential of Cynaroside and Orientin-In Silico (DYRK2) and In Vitro (U87 and Caco-2) Studies.

Luteolin derivates are plant compounds with multiple benefits for human health. Stability to heat and acid hydrolysis and high resistance to (auto)oxidation are other arguments for the laden interest in luteolin derivates today. The present study was designed to compare the in silico and in vitro anti-proliferative potential of two luteolin derivates, luteolin-7-O-glucoside/cynaroside (7-Lut) and luteolin-8-C-glucoside/orientin (8-Lut). In silico investigations were carried out on the molecular target, namely, the human dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) in association with its natural ligand, curcumin (PDB ID: 5ZTN), by CLC Drug Discovery Workbench v. 1.5.1. software and Molegro Virtual Docker (MVD) v. MVD 2019.7.0. software. In vitro studies were performed on two human tumor cell lines, glioblastoma (U87) and colon carcinoma (Caco-2), respectively. Altogether, docking studies have revealed 7-Lut and 8-Lut as effective inhibitors of DYRK2, even stronger than the native ligand curcumin; in vitro studies indicated the ability of both luteolin glucosides to inhibit the viability of both human tumor cell lines, up to 85% at 50 and 100 µg/mL, respectively; the most augmented cytotoxic and anti-proliferative effects were obtained for U87 exposed to 7-Lut (IC50 = 26.34 µg/mL). The results support further studies on cynaroside and orientin to create drug formulas targeting glioblastoma and colon carcinoma in humans.

Potential Benefits of Dietary Plant Compounds on Normal and Tumor Brain Cells in Humans: In Silico and In Vitro Approaches.

Neuroblastoma can be accessed with compounds of larger sizes and wider polarities, which do not usually cross the blood-brain barrier. Clinical data indicate cases of spontaneous regression of neuroblastoma, suggesting a reversible point in the course of cell brain tumorigenesis. Dual specificity tyrosine-phosphorylation-regulated kinase2 (DYRK2) is a major molecular target in tumorigenesis, while curcumin was revealed to be a strong inhibitor of DYRK2 (PBD ID: 5ZTN). Methods: in silico studies by CLC Drug Discovery Workbench (CLC) and Molegro Virtual Docker (MVD) Software on 20 vegetal compounds from the human diet tested on 5ZTN against the native ligand curcumin, in comparison with anemonin. In vitro studies were conducted on two ethanolic extracts from Anemone nemorosa tested on normal and tumor human brain cell lines NHA and U87, compared with four phenolic acids (caffeic, ferulic, gentisic, and para-aminobenzoic/PABA). Conclusions: in silico studies revealed five dietary compounds (verbascoside, lariciresinol, pinoresinol, medioresinol, matairesinol) acting as stronger inhibitors of 5ZTN compared to the native ligand curcumin. In vitro studies indicated that caffeic acid has certain anti-proliferative effects on U87 and small benefits on NHA viability. A. nemorosa extracts indicated potential benefits on NHA viability, and likely dangerous effects on U87.

Combination of Caffeic Acid Phenethyl Ester and Crocin Realign Potential Molecular Markers in U87-MG Glioma Cells.

Background: Glial tumors are the most common primary malignant central nervous system tumors. They are hard to treat, not only because of the deregulation in multiple pathways but also because they are not contained in a well-defined mass with clear borders. The use of a single therapeutic agent to target gliomas has yielded unsatisfactory results. Objective: A combination of molecules targeting multiple pathways may prove to be a better alternative. Methods: The effect of caffeic acid phenethyl ester and crocin on the proliferation and death of U87-MG cells over a concentration range was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. A colony formation assay was used to measure the effect of caffeic acid phenethyl ester and crocin on contact inhibition and anchorage independence ability of U87-MG cells. Furthermore, apoptosis in U87-MG cells was analyzed by propidium iodide assay. Real-time polymerase chain reaction and Western blotting were performed to determine the expression level of p53, epidermal growth factor receptor, and proliferating cell nuclear antigen. Results: Caffeic acid phenethyl ester and crocin when used in combination present an anticancer potential for glioma. These molecules, in combination, inhibit proliferation and induce apoptosis in U87-MG glioma cells. Our results provide evidence that combination treatment realigns the expression paradigm of p53, epidermal growth factor receptor, and proliferating cell nuclear antigen in cotreated U87-MG cells. Conclusions: The combination of caffeic acid phenethyl ester and crocin led to inhibition in glioma cell proliferation and might prove to be an effective adjunct to the therapies in vogue.

The effects of CHF6467, a new mutated form of NGF, on cell models of human glioblastoma. A comparison with wild-type NGF.

CHF6467 is a mutated form of human recombinant nerve growth factor (NGF). The mutation selectively disrupts the binding of NGF to its p75NTR receptor while maintaining the affinity toward TrkA receptor. Because of such different profile of receptor interaction, CHF6467 maintains unaltered the neurotrophic and neuroprotective properties of wild-type NGF but shows reduced algogenic activity.In this study, we investigated the effects of CHF6467 on mortality, proliferation, cell-damage and migration in three human glioblastoma cell lines (U87MG, T98G, LN18), and in the rat astrocytoma C6 cells. Both CHF6467 and wild-type NGF, given in the range 1-50 ng/ml, did not modify cell proliferation, metabolism and migration, as well as the number of live/dead cells.The present in vitro data are predictive of a lack of tumorigenic activity by both wild-type NGF and CHF6467 on these cell types in vivo, and warrant for CHF6467 further clinical development.